The ribosome, as the site where genetic information is translated into polypeptides which subsequently folds into proteins and as the target site for antibiotic action, is an organelle with eminent public health relevance. Recent cryoelectron microscopy reconstructions of the ribosome in the PI's lab have dramatically changed our picture of the translational apparatus, and have provided the first 3D visualizations of tRNA in situ on the ribosome. Both the availability of the cryo-EM envelope (at 25 Angstroms resolution) and the identification of anticodon and peptidyl transferase regions provide a much improved basis for ribosomal RNA modeling. In addition, we show in preliminary experiments that 3D localization of oligo probes is now feasible, furnishing markers that link stretches of RNA sequence directly with regions of the ribosome 3D map. This proposal describes a four-year research plan that seeks to make exhaustive use of these new opportunities in combining medium- resolution modeling of rRNA (following the constraint satisfaction approach) with extensive experimental site localizations. These efforts will be augmented by atomic modeling of functional regions (in collaboration with Dr. Steve Harvey). Specifically, we will separately model the small subunit and large subunit and join the models using experimental constraints (tRNA direct visualization and RNA "bridge" seen in cryo-EM map). This approach will be extended to the eukaryotic ribosome as cryo data become available. We will also develop interactive tools to aid in the building of 3D medium resolution (10-15 Angstroms) models of ribonucleoprotein complexes.